Methods to stimulate biogenic methane production from hydrocarbon-bearing formations

ABSTRACT

The present invention describes methods of stimulating the biogenic production of methane in hydrocarbon-bearing formations. The present application provides various stimulants which, when contacted with a hydrocarbon deposit in situ or ex situ, induce or enhance coalbed methane production.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application No.61/351,709, filed on Jun. 4, 2010, the disclosure of which isincorporated by reference herein in its entirety.

This application is related to U.S. application Ser. No. 12/464,832,filed May 12, 2009, now publication no. US20100047793A1.

BACKGROUND OF THE INVENTION

Coalbed methane (CBM) is a source of natural gas produced eitherbiologically or thermogenically in coal deposits. Biogenic production ofCBM is the result of microbial metabolism and the degradation of coalwith a subsequent electron flow among multiple microbial populations.Thermogenic production of CBM is the result of thermal cracking ofsedimentary organic matter or oil, occurring later in coalification whentemperatures rise above levels at which the methane-producingmicroorganisms can live. In coalbeds, pressure from overlying rock andsurrounding water cause the CBM to bond to the surface of the coal andbe absorbed into the solid matrix of the coal as free gas withinmicropores and cleats (natural fractures in the coal), as dissolved gasin water, as adsorbed gas held by molecular attraction on surfaces ofmacerals (organic constituents that comprise the coal mass), micropores,and cleats in the coal, and as absorbed gas within the molecularstructure of the coal.

Coal is a sedimentary rock with various degrees of permeability, withmethane residing primarily in the cleats. These fractures in the coalact as the major channels to allow CBM to flow. To extract the CBM, asteel-encased hole is drilled into the coal seam, which allows thepressure to decline due to the hole to the surface or the pumping ofsmall amounts of water from the coalbed (dewatering). CBM has very lowsolubility in water and readily separates as pressure decreases,allowing it to be piped out of the well separately from the water. TheCBM is then sent to a compressor station and into natural gas pipelines.

CBM represents a significant portion of the natural gas produced in theUnited States, estimated as providing approximately 10% of the naturalgas supplies, or about 1.8 trillion cubic feet (TCF). Internationalreserves provide enormous opportunity for future CBM production. Amongthe most productive areas is the San Juan Basin, located in Colorado andNew Mexico. Based on such enormous reservoirs of CBM, minimalimprovements in CBM recovery could thus result in significantlyincreased production from a well, and accordingly, a variety of methodsare being developed to improve the recovery of CBM from coal seams.

Purely physical interventions can include optimizing drilling andfracturing methods. Other improvement methods involve the application ofexternal factors directly onto the coalbeds. These include, for example,the injection of gases such as nitrogen (see, e.g., Shimizu et al.,(2007) Molecular characterization of microbial communities in deep coalseam groundwater of northern Japan. Geobiology 5(4):423-433; U.S. Pat.No. 4,883,122) and CO₂ (see, e.g., U.S. Pat. No. 5,402,847); and theinjection of hot fluids such as water or steam (see, e.g., U.S. Pat. No.5,072,990). Various methods are intended to increase the permeability ofthe coalbed seams either physically (see, e.g., U.S. Pat. No. 5,014,788)or chemically (see, e.g., U.S. Pat. No. 5,865,248).

SUMMARY OF THE INVENTION

There remains a need to effectively stimulate biogenic production inhydrocarbon-bearing formations such as coal and to enhance the CBMproductivity of existing wells.

The present invention provides methods and processes for the use ofcompositions comprising stimulants for biogenic production of methane inhydrocarbon-bearing formations. The present invention provides methodsfor tailored interventions, such as the use of compositions comprisingstimulants that can be introduced into an in situ environment to enhancethe biogenic production of methane. The present invention also providesmethods for tailored interventions, such as the use of compositionscomprising stimulants that can be introduced into an ex situ environmentto enhance the biogenic production of methane.

In one embodiment, one or more microorganisms from thehydrocarbon-bearing formation are enriched by selecting for the abilityto grow on coal as the sole carbon source.

In another embodiment, the methods comprise in vitro testing ofcompositions comprising stimulants at more than one concentration tomonitor and optimize methane production in a culture system comprisingat least one microorganism isolated from said hydrocarbon-bearingformation, further wherein said culture system provides coal as the solecarbon source.

At least one microorganism is a bacterial species or an archaeal speciescapable of converting a hydrocarbon to a product selected from the groupconsisting of hydrogen, carbon dioxide, acetate, formate, methanol,methylamine, or any other methanogenic substrate; one or morehydrocarbon-degrading bacterial species, one or more methanogenicbacterial species or one or more methanogenic archaeal species that canconvert substrates to methane.

In one embodiment, the methods are performed with a functional microbialsubcommunity (enrichment) that is developed methods described in Example1 below. The members of the functional microbial subcommunity act inconcert to produce methane; and further wherein said culture systemprovides coal as the sole carbon source.

In an alternative embodiment, the methods are performed with a definedmicrobial assemblage that combines a culture of microorganisms from ahydrocarbon-bearing formation, such that members of said definedmicrobial assemblage act in concert to produce methane; and furtherwherein said culture system provides coal as the sole carbon source.

A hydrocarbon-bearing formation to be treated can be any formationcontaining hydrocarbons. Hydrocarbon-bearing formations include, but arenot limited to: coal, peat, kerogen, oil, tar, heavy oil, oil shale, oilformation, traditional black oil, viscous oil, oil sands and tar sands.In one embodiment, the formation is coal in a coal seam or coalbed. Theterm “coal” as used herein refers to any rank of coal ranging fromlignite to anthracite. The members of the various ranks differ from eachother in the relative amounts of moisture, volatile matter, and fixedcarbon contained in the matrix. The lowest in carbon content, lignite orbrown coal, is followed in ascending order by subbituminous coal orblack lignite (a slightly higher grade than lignite), bituminous coal,semi-bituminous (a high-grade bituminous coal), semi-anthracite (alow-grade anthracite), and anthracite. Coals for use in the presentmethods can be of any rank; representative examples of coal include, butare not limited to, lignite, brown coal, subbituminous coal, bituminouscoal, coking coals, anthracite, and combinations thereof.

In various embodiments, the stimulant involved in the conversion ofhydrocarbon to methane is yeast extract, sulfur, an oxyanion of sulfur(e.g., thiosulfate (S₂O₃), sodium thiosulfate (Na₂S₂O₃), potassiumthiosulfate (K₂S₂O₃), sulfuric acid, disulfuric acid, peroxymonosulfuricacid, peroxydisulfuric acid, dithionic acid, thiosulfuric acid,disulfurous acid, sulfurous acid, dithionus acid or polythionic acid),NH₄Cl, KCl, vanadium, VCl₃, VCl₂, VCl, Na₂SO₃, MnCl₂, Na₂MoO₄, FeCl₃ orNa₂SO₄. In a preferred embodiment, the stimulant is vanadium, VCl₃,VCl₂, VCl, sulfur, thiosulfate or sodium thiosulfate.

The invention provides processes for enhancing biogenic production ofmethane in a hydrocarbon-bearing formation, said method comprisingintroducing a composition comprising a into a hydrocarbon-bearingformation.

In one embodiment, the process introduces the composition comprising thestimulant into the hydrocarbon-bearing formation. In a preferredembodiment, the hydrocarbon-bearing formation is coal.

The invention further provides processes for enhancing biogenicproduction of methane from coal by introducing one or moremicroorganisms, consortiums, functional microbial subcommunities, or aDMA into a coalbed. Microorganisms can be indigenous or exogenous to theformation to be treated. Compositions can include microorganisms thatare naturally-occurring, genetically-engineered, or a combinationthereof. Where more than one population of microorganisms is to beintroduced, one or more populations can be genetically engineered andone or more populations can be genetically unmodified. In suchembodiments, such processes comprise introducing compositions comprisingone or more microorganisms, consortiums, functional microbialsubcommunities, or a DMA into a coalbed together with a compositioncomprising a stimulant.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentapplication can be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the application are utilized, and the accompanyingdrawings of which:

FIGS. 1A and 1B illustrate methane production from coal afterstimulation of a functional microbial subcommunity with a compositioncomprising vanadium (III) chloride (VCl₃) and a composition comprisingsodium thiosulfate (NaS₂O₃), respectively. Each sample was run in fourreplicates; standard error bars are shown for each time point. Week 5methane production is shown for three concentrations of stimulantstested in the presence or absence of coal.

FIGS. 2A and 2B illustrate methane production from coal afterstimulation of a functional microbial subcommunity with a compositioncomprising ammonium chloride (NH₄Cl) and a composition comprising sodiumsulfite (Na₂SO₃), respectively. Each sample was run in four replicates;standard error bars are shown for each time point. Week 5 methaneproduction is shown for three concentrations of stimulants tested in thepresence or absence of coal.

FIGS. 3A and 3B illustrate methane production from coal afterstimulation of a functional microbial subcommunity with a compositioncomprising manganese chloride (MnCl₂) and a composition comprisingsodium molybdic (Na₂MoO₄), respectively. Each sample was run in fourreplicates; standard error bars are shown for each time point. Week 5methane production is shown for three concentrations of stimulantstested in the presence or absence of coal.

FIGS. 4A and 4B illustrate methane production from coal afterstimulation of a functional microbial subcommunity with a compositioncomprising potassium chloride (KCl) and a composition comprising ferrouschloride (FeCl₃), respectively. Each sample was run in four replicates;standard error bars are shown for each time point. Week 5 methaneproduction is shown for three concentrations of stimulants tested in thepresence or absence of coal.

FIG. 5 illustrates methane production from coal after stimulation of afunctional microbial subcommunity with a composition comprising sodiumsulfate (Na₂SO₄). Each sample was run in four replicates; standard errorbars are shown for each time point. Week 5 methane production is shownfor three concentrations of stimulants tested in the presence or absenceof coal.

FIG. 6 illustrates methane production from coal at weeks 1, 3 and 5following stimulation of a functional microbial subcommunity with anintermediate concentration of VCl₃.

FIG. 7 illustrates methane production from coal at weeks 1, 3 and 5following stimulation of a functional microbial subcommunity with anintermediate concentration of NaS₂O₃.

FIG. 8 illustrates a variety of potential enzymatic pathways in theconversion of coal to methane.

FIG. 9 illustrates a process for introducing an external factor such asa stimulant to a coalbed via injected formation water to increasemethane production.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. Many of the techniques andprocedures described or referenced herein are well understood andcommonly employed using conventional methodology by those skilled in theart.

The singular form “a”, “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of cells and reference to “a compound”includes a plurality of compounds, etc.

As used herein, the terms “about” or “approximately” when referring toany numerical value are intended to mean a value of plus or minus 10% ofthe stated value. For example, “about 50 degrees C.” (or “approximately50 degrees C.”) encompasses a range of temperatures from 40 degrees C.to 60 degrees C., inclusive. Similarly, “about 100 mM” (or“approximately 100 mM”) encompasses a range of concentrations from 90 mMto 110 mM, inclusive. All ranges provided within the application areinclusive of the values of the upper and lower ends of the range.

The term “substantially purified”, as used herein, refers to a moleculeseparated from substantially all other molecules normally associatedwith it in its native state. More preferably a substantially purifiedmolecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than 60% free, preferably75% free, more preferably 90% free, and most preferably 95% free fromthe other molecules (exclusive of solvent) present in the naturalmixture. The term “substantially purified” is not intended to encompassmolecules present in their native state.

As used herein, the term “yield” refers to the amount of harvestableproduct, and is normally defined as the measurable produce of economicalvalue of methane. Yield may be defined in terms of quantity or quality.The harvested material may vary from hydrocarbon deposit to hydrocarbondeposit. The term “yield” also encompasses yield potential, which is themaximum obtainable yield. Yield may be dependent on a number of yieldcomponents, which may be monitored by certain parameters. Theseparameters are well known to persons skilled in the art and vary fromdeposit to deposit.

The present invention provides novel methods and processes to stimulatebiogenic methane production in hydrocarbon-bearing formations, such ascoal seams and coalbed methane wells, by stimulating cultivatedmicroorganisms derived from the formation with various amendments. Thepresent application also relates to further stimulating biogenic methaneproduction in a hydrocarbon-bearing formation by exposing the formationby further exposing the formation to one or more microorganisms. Themicroorganisms can be consortiums, isolated cultures, geneticallymodified microorganisms.

The methods of the present invention provide an approach for the use ofstimulants, functional microbial communities, and/or DMAs useful forincreasing biogenic production of methane. Briefly, in the examplesprovided herein, formation water samples were collected from a coalbedmethane well in the San Juan Basin, where previous studies indicated anage of 70 million years resulting from an isolation from the surface andno evidence of subsurface mixing events. The water could be collectedfrom the well head, the separation tank (knock out drum) or reservoirtank as these water samples are the most readily available materials.The water samples containing living microorganisms were then visualizedvia light microscopy, and microorganisms were cultivated using formationwater as mineral base. Cultures of microorganisms were enriched formethane-producing microbes using coal as sole carbon source. Variouscombinations of amendments were tested as stimulants for microbialrespiration. The microbial enrichments (functional functional microbialcommunities) were then screened for methane production using gaschromatography.

The power of the methods of the present invention can be seen in the useof compositions comprising vanadium or thiosulfate to stimulate biogenicproduction of methane from coal.

Sources of Microorganisms and Their Characterization

As used herein, the term “hydrocarbon-bearing formation” refers to anyhydrocarbon source from which methane can be produced, including, butnot limited to, coal, kerogen, peat, oil shales, oil formations, heavyoil, traditional black oils, viscous oil, oil sands and tar sands. Inthe various embodiments discussed herein, a hydrocarbon-bearingformation or even a hydrocarbon-bearing formation environment mayinclude, but is not limited to, coal, coal seam, waste coal, coalderivatives, peat, kerogen, oil formations, oil shale, tar, tar sands,hydrocarbon-contaminated soil, petroleum sludge, drill cuttings, and thelike and may even include those conditions or even surroundings inaddition to oil shale, coal, coal seam, waste coal, coal derivatives,peat, oil formations, tar sands, hydrocarbon-contaminated soil,petroleum sludge, drill cuttings, and the like. In some embodiments, thepresent invention may provide an in situ hydrocarbon-bearing formationsometimes referred as an in situ hydrocarbon-bearing formationenvironment or in situ methane production environment. Embodiments mayinclude an ex situ hydrocarbon-bearing formation sometimes referred toas an ex situ hydrocarbon-bearing formation environment or an ex situmethane production environment. In situ may refer to a formation orenvironment of which hydrocarbon-bearing sources may be in theiroriginal source locations, for example, in situ environments may includea subterranean formation. Ex situ may refer to formations orenvironments where a hydrocarbon-bearing formation has been removed fromits original location and may perhaps even exist in a bioreactor, exsitu reactor, pit, above ground structures, and the like situations. Asa non-limiting example, a bioreactor may refer to any device or systemthat supports a biologically active environment.

Using coal as an exemplary hydrocarbon-bearing formation, there arenumerous sources of indigenous microorganisms that may be playing a rolein the hydrocarbon to methane conversion that can be analyzed. Coal is acomplex organic substance that is comprised of several groups ofmacerals, or major organic matter types, which accumulate in differenttypes of depositional settings such as peat swamps or marshes. Maceralcomposition, and therefore coal composition, changes laterally andvertically within individual coal beds. Once microorganisms areidentified as involved in a conversion step, different functionalmicrobial subcommunities, defined microbial assemblages and/orstimulants identified herein may work better on specific maceral groupsand therefore, each coal bed may be unique in what types ofmicroorganism and stimulant are most efficient at the in situbioconversion of the coal.

There are numerous naturally-occurring microbes that are associated withcoal and other organic-rich sediments in the subsurface. Over time,these microbial species may have become very efficient at metabolizingorganic matter in the subsurface through the process of naturalselection. The relatively quick adaption of bacteria to localenvironmental conditions suggests that microorganisms collected frombasins, or individual coal seams, may be genetically unique. Oncecollected, these microorganisms can be grown in laboratory cultures asdescribed herein to evaluate and determine factors enhancing and/orlimiting the conversion of coal into methane. In some cases, a keynutrient or trace element may be missing, and addition of this limitingfactor may significantly increase methane production. When bacteria aredeprived of nutrients, physiological changes occur, and if the state ofstarvation continues, all metabolic systems cease to function and thebacteria undergo metabolic arrest. When environmental conditions change,the bacteria may recover and establish a viable population again.Therefore, it is possible that some bacteria in organic-rich sedimentshave reached a state of metabolic arrest and the addition of nutrientsis all that is required to activate the population under the presentinvention. By specifically analyzing the effect of various amendments onsuch populations, we can identify compounds that methane productionbeing carried out by one or more members of these microbial populations.

Anaerobic bacteria from a subsurface formation can be collected byseveral different methods that include (1) produced or sampled formationwater, (2) drill cuttings, (3) sidewall core samples (4) whole coresamples, and (5) pressurized whole core samples. Pressurized coresamples may present the best opportunity to collect viable microbialpopulations, but we have found collection of microbial populations fromformation waters has provided a representative sample of the microbialpopulations present. Methanogens are obligate anaerobes, but can remainviable in the presence of oxygen for as much as 24 hours by formingmulticellular lumps. Additionally, anoxic/reducing microenvironments inan oxygenated system can potentially extend anaerobic bacterialviability longer. In some cases, drill cuttings collected and placed inanaerobic sealed containers will contain microorganisms that are capableof converting the coal to methane within a few hours, thereby givingerroneous gas content measurements.

Methods of on-site collection have been optimized to provide optimalrecovery of anaerobic populations of microorganisms therein. The presentinvention involves anaerobic microbial populations previously describedby PCT Application No. PCT/US2008/057919 (WO 2008/116187), and thecultivation of indigenous microorganisms residing in thehydrocarbon-bearing formation environment, such formation water orcoalbed methane wells.

The methods provided herein also afford the opportunity for geneticallyaltering microorganisms. By identifying stimulants that may be used toincrease methane production, microorganisms can be geneticallyengineered to have abilities that can be tied to increased methaneproduction. Selections of microorganisms by the methods described hereinenrich for the ability to efficiently metabolize coal and otherorganic-rich substrates. Various possibilities to enhance methaneproduction from wells comprise introducing compositions comprisingstimulants, microorganisms, defined assemblages of organisms,genetically-modified organisms, or any combinations thereof into theformation.

According to the present methods, a functional microbial community isstimulated to transform hydrocarbons to methane. Microorganismsnaturally present in the formation are preferred because it is knownthat they are capable of surviving and thriving in the formationenvironment, and should provide components of various pathwaysproceeding from hydrocarbon hydrolysis through to methanogenesis.However, this invention is not limited to use of indigenousmicroorganisms. When analyzing enzymatic profiles of indigenousmicroorganisms, it may be advantageous to combine such information withthat of exogenous microorganisms. This information may come from knownmicroorganisms, preferably those that are suitable for growing in thesubterranean formation, and by analogy, have similar potentialprocesses.

The terms “functional microbial community” or “microbial enrichment” asused herein, refers to a culture of more than one microorganism whereinthe community has been developed by culturing a sample under specificconditions. The community may not necessarily remain static over time,but may continue to evolve depending upon nutrient supplements orsubstrates added to the culture.

The term “defined microbial assemblage” or “DMA” as used herein, refersto a culture of more than one microorganism, wherein different strainsare cultured or intentionally combined to convert a hydrocarbon tomethane. The microorganisms of the assemblage are “defined” such that atany point in time we can determine the members of the population by useof genetic methods, such as 16S taxonomy as described herein. The DMAdoes not necessarily remain static over time, but may evolve as culturesflux to optimize hydrocarbon hydrolysis and methane production.Optimally, the DMA is prepared to provide microorganisms harboringstrong capacity to convert hydrocarbon to methane. The DMA may consistof 2 or more microorganisms, in any combination, to provide bacterial orarchael species capable of converting a hydrocarbon to any intermediateleading to the production of methane, and/or any methanogenic species.For example, there may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or more organisms present in a DMA. The members of the DMA actsynergistically to produce methane, amongst themselves, or together withmicroorganisms present in the hydrocarbon-bearing formation.

The term “microorganism” is intended to include bacteria and archaeaorganisms, as well as related fungi, yeasts and molds. It will beunderstood that bacteria and archaea are representative ofmicroorganisms in general that can degrade hydrocarbons and convert theresulting products to methane. The dividing lines between classes ofmicroorganisms are not always distinct, particularly between bacteriaand fungi. It is preferred, therefore, to use the term microorganisms toinclude all microorganisms that can convert hydrocarbons to methane,whatever the commonly used classifications might be. Of thesemicroorganisms, those usually classified as bacteria and archaea are,however, preferred. If exogenous bacteria and archaea are used in themethods described herein, other microorganisms such as fungi, yeasts,molds, and the like can also be used.

The term “anaerobic microorganism” as used herein, refers tomicroorganisms that can live and grow in an atmosphere having less freeoxygen than tropospheric air (i.e., less than about 18%, by mol., offree oxygen). Anaerobic microorganisms include organisms that canfunction in atmospheres where the free oxygen concentration is less thanabout 10% by mol., or less than about 5% by mol., or less than about 2%by mol., or less than about 0.5% by mol.

The term “facultative anaerobes” as used herein, refers tomicroorganisms that can metabolize or grow in environments with eitherhigh or low concentrations of free oxygen.

The conversion of hydrocarbons to methane requires the activeparticipation of methanogens. A “methanogen” as used herein, refers toobligate and facultative anaerobic microorganisms that produce methanefrom a metabolic process. The presence of methanogens within the samplesindicates the high likelihood of in situ methane formation. Methanogensare typically classified into four major groups of microorganisms:Methanobacteriales, Methanomicrobacteria and relatives, Methanopyralesand Methanococcales. All methanogenic microorganisms are believed toemploy elements of the same biochemistry to synthesize methane.Methanogenesis is accomplished by a series of chemical reactionscatalyzed by metal-containing enzymes. One pathway is to reduce CO₂ toCH₄ by adding one hydrogen atom at a time (CO₂-reducing methanogenesis).Another pathway is the fermentation of acetate and single-carboncompounds (other than methane) to methane (acetate fermentation, oracetoclastic methanogenesis). The last step in all known pathways ofmethanogenesis is the reduction of a methyl group to methane using anenzyme known as methyl reductase. As the presence of methyl reductase iscommon to all methanogens; it is a definitive character of methanogenicmicroorganisms. The method for identifying the presence of methanogensis to test directly for the methanogen gene required to produce themethyl reductase enzyme. Alternatively the presence of methanogens canbe determined by comparison of the recovered 16S rDNA against anarchaeal 16S rDNA library using techniques known to one skilled in theart (generally referred to herein as 16S taxonomy).

Classes of methanogens include Methanobacteriales, Methanomicrobacteria,Methanopyrales, Methanococcales, and Methanosaeta (e.g., Methanosaetathermophila), among others. Specific examples of methanogens includeMethanobacter thermoautotorophicus, and Methanobacter wolfeii.Methanogens may also produce methane through metabolic conversion ofalcohols (e.g., methanol), amines (e.g., methylamines), thiols (e.g.,methanethiol), and/or sulfides (e.g., dimethyl sulfide). Examples ofthese methanogens include methanogens from the genera Methanosarcina(e.g., Methanosarcina barkeri, Methanosarcina thermophila,Methanosarcina siciliae, Methanosarcina acidovorans, Methanosarcinamazeii, Methanosarcinafrisius); Methanolobus (e.g., Methanolobusbombavensis, Methanolobus tindarius, Methanolobus vulcani, Methanolobustaylorii, Methanolobus oregonensis); Methanohalophilus (e.g.,Methanohalophilus mahii, Methanohalophilus euhalobius); Methanococcoides(e.g., Methanococcoides methylutens, Methanococcoides burtonii); and/orMethanosalsus (e.g., Methanosalsus zhilinaeae). They may also bemethanogens from the genus Methanosphaera (e.g., Methanosphaerastadtmanae and Methanosphaera cuniculi, which are shown to metabolizemethanol to methane). They may further be methanogens from the genusMethanomethylovorans (e.g., Methanomethylovorans hollandica, which isshown to metabolize methanol, dimethyl sulfide, methanethiol,monomethylamine, dimethylamine, and trimethylamine into methane).

As described herein, it is a feature of the present embodiments thatmicrobial communities obtained from a variety of environmental samplesare amenable to study using genomic tools as provided herein; inaddition, microbial populations can be cultivated and optionallyisolated and/or enriched in the laboratory using invention methods. Byapplying these approaches at the genomic level, and by specificallycharacterizing the enzymatic profiles of microorganisms involved in theconversion of hydrocarbons to methane, it is possible to develop afundamental understanding of the metabolism of the microbial communitiesand, more specifically, the methanogenic degradation of coal in theformation water and coal seams. As such, we are then able to elucidatethe ecological niche of each population and ultimately developstimulants, functional microbial subcommunities and/or DMAs that couldyield an enhancement in the biological methane production.

According to the present methods, microorganisms present in thehydrocarbon-bearing formation environment (indigenous microorganisms)are stimulated or modulated to transform hydrocarbons to methane.Microorganisms naturally present in the formation are preferred becauseit is known that they are capable of surviving and thriving in theformation environment. However, this invention is not limited to use ofindigenous microorganisms. Exogenous microorganisms suitable for growingin the subterranean formation may be identified and such microorganismsintroduced into the formation by known injection techniques before,during, or after practicing the process of this invention. For example,if the formation contains only two microorganisms of a desiredthree-component consortia, then the missing microorganisms, or astimulant for such a microorganism could be injected into the formation.Microorganisms, indigenous or exogenous, may also be recombinantlymodified or synthetic organisms.

Stimulants

The term “stimulant” as used herein refers to any factor that can beused to increase or stimulate the biogenic production of a metabolicproduct with increased hydrogen content from a hydrocarbon material.Metabolic products with increased hydrogen content include, but are notlimited to, methane, hydrogen, acetate, formate, butyrate, propionate,substituted and un-substituted hydrocarbons, such as ethers, aldehydes,ketones, alcohols, organic acids, amines, thiols, sulfides, anddisulfides, among others, substituted and unsubstituted, mono- andpoly-aromatic hydrocarbons, and the like. In one embodiment, themetabolic product with increased hydrogen content is methane.

A stimulant can be a substrate, reactant or co-factor for a pathway thatis involved in the conversion of a hydrocarbon to methane. The functionof the stimulant is to boost existing production by increasing the levelof activity or growth of a microorganism, or to increase, decrease ormodulate by any means the enzymatic activity of an enzyme involved in apathway involved in the conversion of a hydrocarbon to methane in orderto optimize the end production of methane from the hydrocarbon-bearingformation.

Stimulants may provide for enhancement, replacement, or addition of anynutrient that is not optimally represented or functional in thehydrocarbon-bearing environment. The goal is to optimize and/or completeof the pathway from hydrocarbon to methane. Generally this requiresrepresentation of microorganisms that are capable of converting ahydrocarbon to a product such as hydrogen, carbon dioxide, acetate,formate, methanol, methylamine or any other methanogenic substrate.Microorganisms include those capable of low rank coal hydrolysis, coaldepolymerization, anaerobic or aerobic degradation of polyaromatichydrocarbons, homoacetogenesis, and methanogenesis (includinghydrogenotrophic or CO₂ reducing and acetoclastic), and any combinationsthereof to achieve conversion of a hydrocarbon to methane.

Examples of stimulants include, for example, yeast extract, sulfur, anoxyanion of sulfur (e.g., thiosulfate (S₂O₃), sodium thiosulfate(Na₂S₂O₃), potassium thiosulfate (K₂S₂O₃), sulfuric acid, disulfuricacid, peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid,thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid orpolythionic acid), NH₄Cl, KCl, vanadium, VCl₃, VCl₂, VCl, Na₂SO₃, MnCl₂,Na₂MoO₄, FeCl₃ or Na₂SO₄. In a preferred embodiment, the stimulant isvanadium or thiosulfate.

Incorporation of Stimulants to Increase Methane Production

The methods and processes of the present invention can be readily usedfor field applications and the enhancement of in situ or ex situ methaneproduction from any hydrocarbon-bearing formation such as coal. Thereare several methods or combination of injection techniques that areknown in the art that can be used in situ. Stimulants, functionalmicrobial subcommunities, DMAs, and/or microorganisms can be injecteddirectly into the fractures in the formation. The stimulant componentsare to be injected as a composition in an aqueous solution such as, butnot limited to, formation water, water or media. Fracture orientation,present day in situ stress direction, reservoir (coal and/or shale)geometry, and local structure are factors to consider. For example,there are two major networks (called cleats) in coal beds, termed theface cleat and butt cleat system. The face cleats are often morelaterally continuous and permeable, whereas the butt cleats (which formabutting relationships with the face cleats) are less continuous andpermeable. During the stimulation of coal bed methane wells, the inducedfractures intersect the primary face cleats that allow greater access tothe reservoir. However, when the present day in situ stress direction isperpendicular the face cleats, then stress pressure closes the facecleats thereby reducing permeability, but at the same time in situpressures increase permeability of the butt cleats system. Under theseconditions, induced fractures are perpendicular to the butt cleatdirection, providing better access to the natural fracture system in thereservoir. The geometry of the injection and producing wells, andwhether or not horizontal cells are used to access the reservoir, dependlargely upon local geologic and hydrologic condition.

The objective of hydraulic fracture stimulation of coal bed methane, asin conventional oil and gas wells, is to generate an induced fracturenetwork that connects with the naturally occurring fracture network ofthe reservoir. Stimulants, functional microbial subcommunities, DMAs,and/or microorganisms can be introduced into the naturally-occurring andartificially-induced fractures under pressure to drive the mixture intonaturally-occurring fractures deep into the reservoir to maximizebioconversion rates and efficiency. During fracture stimulation ofreservoirs, sand proppant and various chemicals may be pumped into theformation under high pressure through a drill rig.

Stimulants, functional microbial subcommunities, DMAs, and/ormicroorganisms may be injected into the reservoir at the same time asfracture stimulation and/or after the hydraulic fractures are generated.Most in situ microbial applications are expected to occur after fracturestimulation and removal of completion fluids when subsurface anaerobicconditions are reestablished. However, under simultaneous in situmicrobial and fracture stimulation, the use of stimulation fluids underanoxic or suboxic conditions is preferred so that anaerobic conditionsin the reservoir are maintained, or can be readily attained afterstimulation. The injection of aerobic bacteria during simultaneousstimulation would result in the rapid consumption of oxygen and returnto anaerobic conditions.

In some cases, pretreatment fluids that modify the coal, carbonaceousshale, or organic-rich shale for bioconversion may be used with thefracture fluids. However, the preferred method for encouraging in situbioconversion of organic matter is to inject compositions comprisingstimulants, functional microbial subcommunities, DMAs, and/ormicroorganisms under pressure and anaerobic conditions after hydraulicfracture stimulation and subsequent flushing of the well.

Stimulants, functional microbial subcommunities, DMAs, and/ormicroorganisms can be introduced by re-introduction of the formationwater to the subsurface as depicted in FIG. 9. Briefly, methane andformation water are pumped from the well casing 1 into the separationtank 2 (also known as the knock out drum) to remove the gas from thewater. The formation water is stored in the reservoir tank 3, from whichit can be forwarded to a consolidation station or directed forre-injection to the subsurface. Stimulants, functional microbialsubcommunities, DMAs, and/or microorganisms can then be added to thepreparation tank 4 and mixed with the recovered formation water. Acompressor 5 or pressurized system can then be used to introduce thestimulants, functional microbial subcommunities, DMAs, and/ormicroorganisms in the formation water to the subsurface.

The introduction of compositions comprising stimulants, functionalmicrobial subcommunities, DMAs, and/or microorganisms, or the deliveryof gases, liquids, gels or solids can provide an environment suitablefor enhanced methane, including strains capable of aerobic degradationof hydrocarbons. For example, in an exemplary embodiment an inoculumcomposed of the suitable strains such as described herein at a cellnumber of 10⁷ cells per ml can be mixed with a gel composed of organicsubstrates such as glycerol than can be used as nutrients stimulatinggrowth through fermentation and secretion of metabolites, includinghydrogen, that can be used by methanogens. Once the gel has beenassimilated, it will slowly release the optimal amounts of stimulantthat will be used by the strains with the capacity for hydrocarbondegradation. These amendments and resulting metabolism can stimulate theelectron flow to methane producing a higher amount and yield compared tocontrol wells in the same seam that are not intervened. This isparticularly advantageous for strains with the capacity to growaerobically or anaerobically and can adapt their metabolism forhydrocarbon degradation. In a separate embodiment, an aqueouscomposition (e.g., formation water, milliΩ water, buffered water, etc.)containing one or more stimulants is injected in a well in order todispense stimulants needed for conversion of a hydrocarbon to methanereactions. One or more additional elements can be further added to theaqueous composition; such further elements include, for example, one ormore of vitamins, trace elements, minerals, or a combination thereof.Exemplary additions include, but are not limited to, Wolfe's vitaminsolutions, Wolfe's trace elements, trace element solution SL-7, traceelement solution SL-10, etc. The concentration of such additionalnutrients can be empirically optimized to the material to be treated andthe conditions of the treatment (e.g., in situ vs. ex situ treatmentconditions).

In an alternative embodiment, a particle-based method can be used todistribute compositions comprising stimulants, functional microbialsubcommunities, DMAs, and/or microorganisms (collectively, theintervention agents) during a fracturing process. The goal is tointroduce these interventions in order to produce an enhancement ofmethane production. A delivery system injects the agents deep into thewell fissures and enables a time-released deployment. For example, thewell intervention agent can be formulated as either a time-releasedcoating over the sand grains used in the fracturing process or as hardparticles which slowly dissolve with time; the size is envisioned asroughly the same as the sand grains used in the fracturing process, andcould be mixed together before added to the guar gum solution known asthe proppant. In either format, once the proppant and particles arepumped into the well and pressured, the coated sand grains or hardparticles mixed with the sand are pressure-injected in the wellfractures, keeping them open to facilitate gas or oil release. Since theintervention agents are formulated in a time-release manner notdissimilar to some pharmaceutical agents, the compounds and/or microbeswould dissolve slowly and diffuse into the surrounding formation waterand into the coal cleats (or fine rock cracks in the case of oil) whereadhered bacteria presumably reside. In this fashion, the dissolvingagents continuously stimulate the biogenic conversion of coal tomethane. The formulations could be fashioned to release the interventionagent over a period of hours, days, weeks or months in order to optimizethe methane stimulation process. The coatings or particles could beprepared in the absence of oxygen in order to maintain the viability ofstrict anaerobic microbes, or they could also harbor gases whichstimulate methane production.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and embodimentswill be apparent upon review of this disclosure. The following examplesare offered to illustrate, but not limit, the disclosed embodiments.

EXAMPLES Example 1 Sampling and Enrichment of Methane-ProducingMicroorganisms from a Coalbed Methane Well (Upflow Reactor 2 L-245)

An inoculum containing a functional methane producing community enrichedfrom formation water was collected from the San Juan Basin.

Formation water was collected from a coalbed methane well located in theSan Juan Basin, Colo., USA. The water was then filtered with a series ofsterile sieves from 1 mm to 45 μm to remove large pieces of coal andoils that came with the formation water. Subsamples were transferredinto a 2 L plastic bottle and a 1 L sterile glass bottle. The glassbottle sample was sparged with N₂ using a portable tank and a glasspipette and then sealed with a sterile butyl stopper. Both bottles weretransferred to the laboratory in less than 12 hours and the 2 L volumewas sterilized by filtration using a 0.2 micron sieve.

Sterile filtered formation water was used as the base for a growthmedium. This base was supplemented with 10 ml/L each of trace metal andvitamin solutions and 200 μg sodium resazurin. A 1 L volume of thissolution was sparged with N₂ gas for 20 minutes, then transferred intoan anoxic glove box and sterile-filtered through a 0.2 micron sieve. Theresulting sterile solution was then dispensed in 5 ml volumes intoHungate tubes containing 0.5 g coal, the tubes were sealed with screwcaps over butyl rubber septa and removed from the glove box.

An electron acceptor stock solution was made by combining 7 g of sodiumsulfate and 1.7 g of sodium nitrate in a serum bottle and adding 50 mlof freshly boiled water. This solution was then immediately sparged for15 minutes with N₂ gas, capped with a butyl rubber stopper, sealed withan aluminum crimp seal and autoclaved at 121° C. for 20 minutes.

A yeast extract stock solution was made by combining 2.5 g yeast extractand 50 ml of freshly boiled water in a serum bottle. This solution wasthen immediately sparged for 15 minutes with N₂ gas, capped with a butylrubber stopper, sealed with an aluminum crimp seal and autoclaved at121° C. for 20 minutes.

To create the primary enrichment culture, an anoxic Hungate tube withcoal and base medium was supplemented with 0.05 ml of the electronacceptor and yeast extract stock solutions using N₂ sparged syringes andneedles and inoculated with 1 ml of anoxic formation water. The primaryenrichment was incubated at 50° C., sampled occasionally for headspacegases and eventually found to produce 6.65% methane after 6 weeks. Theenrichment was then transferred by adding 1 ml to an identical,previously uninoculated, Hungate tube using an N₂ sparged syringe andneedle. This tube, the secondary enrichment, was found to have 10.2%headspace methane after four weeks and transferred again in the samemanner to create a tertiary enrichment.

The tertiary enrichment culture was maintained in an anaerobic reactorsystem as described by (Lettinga, G. 1995. Anaerobic digestion andwastewater treatment systems. Antonie van Leeuwenhoek 67:3-28). Thereactor was a 2000-mL laboratory-scale glass reactor equipped with aheat jacket equilibrated to 50° C. The reactor was fitted with ports totake liquid and gas samples close to the reactor outlet. The effluentwas recycled in a relationship of 4:1 (80% recirculation) relative tothe inlet flow. Production of methane was monitored by GC-FID. Beforestart-up of the reactor system was filled with coal and autoclaved (30minutes) and sparged with anaerobic gas (80% N₂/20% CO₂). The reactorwas started DMY-media with a hydraulic retention time (HRT) of 48 h.

Example 2 Stimulation of Methane Production

The availability of an inoculum of a functional microbial subcommunitycapable of producing methane from coal in vitro prompted laboratoryexperiments where various stimulants were tested for their effect onmethane production.

Materials

Below is the detailed description of growth media composition, coalsample used and microbial inoculum for the batch experiments.

Inoculum 2L-245 (10% vol./vol.) Growth Medium DM Incubation Temp 50° C.Head-space atmosphere 20% CO₂/Bal. N₂ Coal Sterility Autoclave 30′Liquid

Additives/Stimulants:

The experiments were conducted at three different levels of additives(0.5×, 1× and 2×): additives (stimulants) were added in the followingthree concentrations.

Concentration (μM) Additive/Stimulant High Medium Low KCl 8000 4000 2000FeCl₃ 74 37 18.5 Na₂MoO₄ 10 5 2.5 MnCl₂ 10 5 2.5 VCl₃ 10 5 2.5 Na₂SO₄500 250 125 Na₂S₂O₃ 200 100 50 NH₄Cl 21 10 5 Na₂SO₃ 500 250 125

Media Preparations

DM media: the media composition in milli-Q water contains 1.2 g/L NaCl,0.4 g/L MgCl₂×6 H₂O, 0.2 g/L CaCl₂×2 H₂O, 0.3 g/L NH₄Cl, 0.3 g/L KCl,2.4 g/L NaHCO₃, 0.25 g/L Na₂S, and 0.2 g/L K₂HPO₄ (Dibasic), 10 ml/L ofWolfe's trace elements and 10 ml/L of Wolfe's vitamins (as describedbelow). After mixing, the solution is sparged with a N₂/CO₂ mixture(80%/20%) to make the solution anaerobic.

DMY media: DM media composition in milli-Q water is made as describedabove with the addition of 0.5 g/L yeast extract.

DMSC media: DM media in milli-Q water is made as described above withthe addition of 100 g/L sterile, anaerobic, sub-bituminous coal from theSan Juan Basin.

DMSCY media: DM media in milli-Q water is made as described above withthe addition of 100 g/L sterile, anaerobic, sub-bituminous coal from theSan Juan Basin and 0.5 g/L yeast extract.

Trace Element and Vitamin Solutions (Concentration in MilliQ Water)

Wolfe's Trace elements (100X) EDTA 0.5 g/L MgSO₄•7H₂O   3 g/L MnSO₄•H₂O0.5 g/L NaCl   1 g/L FeSO₄•7H₂O 0.1 g/L Co(NO₃)₂•6H₂O 0.1 g/LCaCl₂(anhydrous) 0.1 g/L ZnSO₄•7H₂O 0.1 g/L CuSO₄•5H₂O 0.01 g/L 

Wolfe's Trace elements (100X) AlK(SO₄)₂(anhydrous) 0.01 g/L H₃BO₃ 0.01g/L Na₂MoO₄•2H₂O 0.01 g/L Na₂SeO₃(anhydrous) 0.001 g/L  Na₂WO₄•2H₂O 0.01g/L NiCl₂•6H₂O 0.02 g/L A one liter solution is made: EDTA is addedfirst to the milliQ water and each element is added individuallythereafter. The solution is sparged with 80% N₂/20% CO₂ and added tosamples anaerobically.

Wolfe's vitamins (100X) Folic acid 2 mg/L Pyridoxine hydrochloride 10mg/L  Riboflavin 5 mg/L Biotin 2 mg/L Thiamine 5 mg/L Nicotinic acid 5mg/L Calcium pantothenate 5 mg/L Vitamin B12 0.1 mg/L   p-Aminobenzoicacid 5 mg/L Thioctic acid 5 mg/L Monopotassium phosphate 900 mg/L 

Coal

The coal used in these experiments originated from the San Juan Basin(BHP Billiton, Tex.). The coal was stored, pulverized and dry-sievedunder anaerobic conditions (>99.5% N₂). Coal used in the experimentsdescribed herein had a particle size of 150 μm to 250 μm.

Methods

Each reaction tube contained a 10% volume/volume sample from the upflowreactor described above in Example 1. Each stimulant was tested in thepresence or absence of coal. The experiments were conducted at threedifferent concentrations of additives (0.5× (low), 1× (medium), and 2×(high)) as described above. Each sample type was conducted in 4replicates. Samples were incubated anaerobically for 5 weeks at 50° C.and headspace samples were taken at weeks 1, 3 and 5. Control sampletypes were media alone, media plus coal, media plus yeast extract andmedia plus coal and yeast extract.

At each time point, headspace measurements were taken using gaschromatography (microGC 3000A, Agilent Technologies, Inc.). Tubes weresampled for H₂, CO₂ and CH₄ at weeks 1, 3 and 5. Each experiment wasperformed in quadruplicate. Methane at time 0 was assumed to be 0%.Although methane production was not measured at time 0 in thisexperiment, multiple experiments have been run in prior experiments, andin each instance, methane production was 0%.

Results

FIGS. 1-5 show the results of stimulation of the culture system withthree concentrations of various additives at week 5. FIGS. 6 and 7 showthe results of stimulation of the functional microbial subcommunity.

Methane production from each additive experiment is provided in theTables below.

TABLE 1 Control samples Week RepA RepB RepC Rep D Average DM 1 0 0 0 00.00 3 0 0 0 0 0.00 5 0.01 0.01 0.01 0.01 0.01 DMSC 1 0.02 0 0.02 0.020.01 3 0 0 0 0.08 0.02 5 0.08 0.053 0.25 2.98 0.84 DMY 1 0.19 0.22 0.200.23 0.21 3 0.69 0 0.64 0.75 0.52 5 0.71 0.67 0.72 0.77 0.72 DMSCY 10.28 0.28 0.26 0.28 0.27 3 0.73 1.01 1.02 1.07 0.96 5 1.26 1.30 1.231.31 1.28

TABLE 2 VCl₃ Week RepA RepB RepC Rep D Average DMY 1 0.22 0.20 0.21 0.210.21 2.5 μM VCl₃ 3 0.75 0.69 0.72 1.10 0.81 5 0.70 0.66 0.64 0.69 0.67DMSCY 1 0.41 0.32 0.27 0.31 0.33 2.5 μM VCl₃ 3 1.70 1.45 1.12 1.18 1.365 2.93 2.40 1.37 1.50 2.05 DMY 1 0.21 0.20 0.20 0.20 0.20 5 μM VCl₃ 30.78 0.69 0.67 0.80 0.71 5 0.75 0.70 0.68 0.67 0.70 DMSCY 1 0.30 0.260.25 0.26 0.27 5 μM VCl₃ 3 1.51 1.40 1.21 1.88 1.50 5 2.41 2.28 1.953.13 2.44 DMY 1 0.21 0.25104 0.19 0.22 0.22 10 μM VCl₃ 3 0.85 0.7495070.70 0.78 0.77 5 0.76 0.82 0.70 0.77 0.76 DMSCY 1 0.29 0.25 0.24 0.240.25 10 μM VCl₃ 3 1.15 1.04 1.04 1.01 1.06 5 1.49 1.31 1.30 1.33 1.36

TABLE 3 Na₂SO₄ Week RepA RepB RepC Rep D Average DMY 1 0.24 0.21 0.200.19 0.21 125 μM Na₂SO₄ 3 0 0.71 0.69 0.22 0.40 5 0.78 0.65 0.62 0.170.55 DMSCY 1 0.35 0.32 0.29 0.34 0.33 125 μM Na₂SO₄ 3 1.164 1.07 1.031.07 1.08 5 1.30 1.27 1.20 1.26 1.26 DMY 1 0.24 0.20 0.24 0.24 0.23 250μM Na₂SO₄ 3 1.11 0.70 0.82 0.33 0.74 5 0.92 0.71 0.83 0.31 0.69 DMSCY 10.26 0.24 0.29 0.29 0.27 250 μM Na₂SO₄ 3 1.21 1.01 1.89 1.45 1.39 5 0.511.63 0.94 1.85 1.23 DMY 1 0.21 0.21 0.20 0.19 0.205 500 μM Na₂SO₄ 3 0.710.45 0.67 0.19 0.51 5 0.70 0.71 0.69 0.22 0.58 DMSCY 1 0.27 0.24 0.240.24 0.25 500 μM Na₂SO₄ 3 1.11 1.08 1.02 1.38 1.15 5 1.39 1.28 1.29 1.321.32

TABLE 4 Na₂S₂O₃ Week RepA RepB RepC Rep D Average DMY 1 0.24 0.24 0.220.23 0.23 50 μM Na₂S₂O₃ 3 0.75 0.66 0.73 0.74 0.72 5 0.75 0.68 0.68 0.720.70 DMSCY 1 0.30 0.29 0.29 0.27 0.29 50 μM Na₂S₂O₃ 3 1.16 1.02 1.041.09 1.08 5 1.47 1.32 1.36 1.34 1.37 DMY 1 0.21 0.19 0.21 0.23 0.21 100μM Na₂S₂O₃ 3 0.70 0.69 0.34 0.74 0.62 5 0.71 0.76 0.75 0.77 0.75 DMSCY 10.24 0.22 0.25 0.26 0.24 100 μM Na₂S₂O₃ 3 1.32 1.18 1.28 1.01 1.20 51.81 1.58 1.87 1.30 1.64 DMY 1 0.18 0.18 0.17 0.17 0.18 200 μM Na₂S₂O₃ 30.66 0.63 0.68 0.63 0.65 5 0.70 0.66 0.63 0.64 0.66 DMSCY 1 0.20 0.170.17 0.18 0.18 200 μM Na₂S₂O₃ 3 1.18 0.85 0.82 0.93 0.95 5 1.14 1.131.11 1.27 1.16

TABLE 5 NH₄Cl Week RepA RepB RepC Rep D Average DMY 1 0.26 0.27 0.270.27 0.26 5 mM NH₄Cl 3 0.74 0.68 0.67 0.74 0.71 5 0.69 0.65 0.62 0.690.66 DMSCY 1 0.35 0.27 0.31 0.30 0.31 5 mM NH₄Cl 3 1.24 1.03 1.13 1.031.11 5 1.67 1.41 1.48 1.43 1.50 DMY 1 0.22 0.21 0.15 0.22 0.20 10 mMNH₄Cl 3 0.73 0.61 0.21 0.73 0.57 5 0.76 0.70 0.17 0.70 0.58 DMSCY 1 0.250.25 0.22 0.28 0.25 10 mM NH₄Cl 3 1.22 1.43 1.41 1.14 1.30 5 1.98 2.702.67 1.54 2.22 DMY 1 0.23 0.22 0.23 0.21 0.22 20 mM NH₄Cl 3 0.80 0.780.76 0.76 0.77 5 0.80 0.79 0.76 0.77 0.78 DMSCY 1 0.24 0.22 0.21 0.220.22 20 mM NH₄Cl 3 1.30 20 1.08 1.15 1.18 5 1.63 1.48 1.37 1.36 1.46

TABLE 6 Na₂SO₃ Week RepA RepB RepC Rep D Average DMY 1 0.20 0.20 0.210.22 0.21 125 μM Na₂SO₃ 3 0.71 0.68 0.69 0.75 0.71 5 0.66 0.69 0.66 0.710.68 DMSCY 1 0.26 0.24 0.25 0.32 0.27 125 μM Na₂SO₃ 3 1.04 0.97 0.991.06 1.02 5 1.21 1.22 1.25 1.31 1.25 DMY 1 0.14 0.15 0.13 0.15 0.14 250μM Na₂SO₃ 3 0.67 0.63 0.65 0.72 0.67 5 0.62 0.59 0.62 0.65 0.62 DMSCY 10.23 0.18 0.19 0.20 20 250 μM Na₂SO₃ 3 1.04 1.02 1.06 1.18 1.07 5 1.261.24 1.45 1.74 1.42 DMY 1 0.09 0.08 0.08 0.10 0.09 500 μM Na₂SO₃ 3 0.710.64 0.64 0.74 0.68 5 0.83 0.68 0.68 0.76 0.74 DMSCY 1 0.14 0.13 0.140.15 0.14 500 μM Na₂SO₃ 3 1.01 0.98 1.07 1.04 1.04 5 1.34 1.26 1.37 1.411.35

TABLE 7 Control samples Week RepA RepB RepC Rep D Average DM only 1 0.000.00 0.00 0.00 0.00 3 0.00 0.00 0.00 0.00 0.00 5 0.00 0.00 0.00 0.000.00 DMSC 1 0.02 0.03 19.05 0.04 4.78 3 0.00 0.00 0.00 0.00 0.00 5 0.170.25 0.056 0.11 0.15 DMY 1 0.41 0.37 0.35 0.42 0.38 3 1.13 1.00 0.991.09 1.05 5 1.11 0.98 0.98 6 1.03 DMSCY 1 0.53 0.41 0.38 0.49 0.45 33.82 1.37 1.30 1.47 1.99 5 4.70 1.71 1.47 1.65 2.38

TABLE 8 MnCl₂ Week RepA RepB RepC Rep D Average DMY 1 0.40 0.32 0.350.36 0.36 2.5 μM MnCl₂ 3 0.96 0.96 0.93 0.94 0.95 5 0.94 0.87 0.91 0.910.91 DMSCY 1 0.36 0.46 0.41 0.42 0.41 2.5 μM MnCl₂ 3 1.40 1.70 1.41 1.501.50 5 1.59 1.90 1.59 1.73 1.70 DMY 1 0.34 0.33 0.34 0.36 0.34 5.0 μMMnCl₂ 3 0.92 0.90 0.90 0.94 0.92 5 0.89 0.86 0.85 0.88 0.87 DMSCY 1 0.240.51 0.49 0.52 0.44 5.0 μM MnCl₂ 3 1.51 1.55 1.53 1.64 1.56 5 1.94 1.811.70 1.85 1.82 DMY 1 0.39 0.38 0.35 0.39 0.36 10 μM MnCl₂ 3 1.00 1.051.04 1.01 1.02 5 0.89 1.03 0.96 1.01 0.97 DMSCY 1 0.44 0.41 0.41 0.430.42 10 μM MnCl₂ 3 1.45 1.49 1.43 1.48 1.46 5 1.63 1.82 1.71 1.70 1.72

TABLE 9 Na₂MoO₄ Week RepA RepB RepC Rep D Average DMY 1 0.38 0.36 0.380.35 0.37 2.5 μM Na₂MoO₄ 3 1.00 0.98 0.96 0.82 0.94 5 1.01 0.95 0.970.82 0.94 DMSCY 1 0.43 0.37 0.36 0.44 0.40 2.5 μM Na₂MoO₄ 3 1.43 1.101.14 1.41 1.27 5 1.61 1.32 1.32 1.57 1.45 DMY 1 0.25 0.27 0.30 0.33 0.295.0 μM Na₂MoO₄ 3 0.77 0.85 0.79 0.91 0.83 5 0.76 0.78 0.74 0.87 0.79DMSCY 1 0.39 0.39 0.28 0.31 0.34 5.0 μM Na₂MoO₄ 3 1.13 1.09 1.14 1.401.19 5 1.39 1.31 1.29 1.63 1.41 DMY 1 0.30 0.26 0.30 0.32 0.29 10 μMNa₂MoO₄ 3 0.85 0.71 0.81 0.90 0.82 5 0.77 0.72 0.74 0.83 0.77 DMSCY 10.43 0.43 0.47 0.44 0.44 10 μM Na₂MoO₄ 3 1.03 1.15 1.14 1.07 1.10 5 1.111.14 1.15 1.13 1.13

TABLE 10 KCl Week RepA RepB RepC Rep D Average DMY 1 0.29 0.30 0.32 0.290.30 2.0 mM KCl 3 0.99 0.79 0.78 0.70 0.81 5 0.78 0.75 0.79 0.75 0.77DMSCY 1 0.35 0.30 0.32 0.39 0.34 2.0 mM KCl 3 1.12 1.25 1.04 1.16 1.14 51.32 1.49 1.30 1.47 1.39 DMY 1 0.23 0.23 0.22 0.24 0.23 4.0 mM KCl 30.24 0.26 0.26 0.29 0.26 5 0.23 0.26 0.26 0.40 0.29 DMSCY 1 0.44 0.430.43 0.46 0.4 4.0 mM KCl 3 1.35 1.46 1.60 1.57 1.50 5 1.59 1.59 1.871.70 1.69 DMY 1 0.200534 0.203707 0.194967 0.206776 0.201496 8.0 mM KCl3 0.221062 0.211906 0.220571 0.217698 0.217809 5 0.217698 0.2081930.223103 0.211229 0.215056 DMSCY 1 0.387142 0.336361 0.421862 0.4290470.393603 8.0 mM KCl 3 1.345807 1.300728 1.359635 1.303668 1.327459 51.564226 1.477874 1.556977 1.648504 1.561895

TABLE 11 FeCl₃ Week RepA RepB RepC Rep D Average DMY 1 0.42 0.43 0.380.42 0.41 18.5 μM FeCl₃ 3 1.01 1.03 0.94 1.03 1.00 5 0.96 1.04 0.93 1.050.99 DMSCY 1 0.40 0.41 0.46 0.42 0.42 18.5 μM FeCl₃ 3 1.47 1.38 1.401.28 1.38 5 1.69 1.61 1.57 1.47 1.59 DMY 1 0.33 0.31 0.30 0.29 0.31 37.0μM FeCl₃ 3 0.88 0.89 0.86 0.82 0.86 5 0.83 0.86 0.79 0.75 0.81 DMSCY 10.47 0.35 0.35 0.43 0.40 37.0 μM FeCl₃ 3 1.20 1.33 1.14 1.47 1.28 51.612 1.44 1.26 1.53 1.46 DMY 1 0.43 0.37 0.36 0.29 0.36 74 μM FeCl₃ 30.84 0.89 0.85 1.01 0.90 5 0.80 0.83 0.81 0.94 0.85 DMSCY 1 0.44 0.060.42 0.53 0.36 74 μM FeCl₃ 3 1.31 0.05 1.30 1.94 1.15 5 1.51 0.06 1.482.26 1.33

VCl₃: A statistically significant positive effect on the production ofmethane was observed at the intermediate concentration of VCl₃ (P=0.0084at 5 μM VCl₃, P=0.0771 at 2.5 μM VCl₃); methane production was onlyobserved in the presence of coal (FIG. 1A).

Sodium thiosulfate: A statistically significant positive effect on theproduction of methane was observed at the intermediate concentration ofsodium thiosulfate; P=0.079); methane production was only observed inthe presence of coal. (FIG. 1B).

NH₄Cl: A statistically significant positive effect on the production ofmethane was observed at the intermediate concentration of NH₄Cl(P=0.0007); methane production was only observed in presence of coal(FIG. 2A).

Molybdate (Na₂MoO₄): There was a negative correlation in methaneproduction when molybdate was added to the cultures (R²=0.69). Effectswith or without coal were not statistically significantly different(P=0.063) (FIG. 3B).

KCl: A decreasing trend in methane production was observed at week 5 inKCL-containing samples. KCl in the absence of coal (DMY) greatlydecreased methanogenesis at >2 mM (FIG. 4A).

Overall, with the exception of KCL, trends with increasing or decreasingconcentrations of methane production were not observed (see, e.g., FIGS.2, 3, 4B and 5).

Thus, vanadium and thiosulfate compositions were observed to increasemethane production using the present methods.

Example 3 Stimulation of Methane Production by Vanadium in CulturesUtilizing Formate

Formate is a likely product of coal matrix degradation and acts asubstrate for methanogenic microbes.

The anaerobic reactor system described above in Example 1 containsformate-utilizing, methane-producing species in the enrichment culture.Methanogens in the culture were previously identified by 16sRNAsequencing.

A 10% vol./vol. inoculum was taken from the anaerobic reactor systemdescribed above in Example 1, and the effect of vanadium on methaneproduction by methanogens utilizing formate as a carbon and energysource was determined.

Cultures, established with DM media (prepared as described above)containing 50 mM formate as the sole carbon and energy source, weresub-cultured with the same media (in the absence of vanadium) or mediasupplemented with either 5 or 10 μM VCl₃. Methane production wasmeasured after 48 hours of growth at 50° C.

Replicate Cultures Methane (% (μM VCl₃ added in final concentration) inhead space volume of 21 ml) none 0.7 none 0.5  5 2.0  5 1.2 10 1.9 101.4

The VCl₃-supplemented cultures showed an average of approximately 2-foldgreater methane formation than cultures without VCl₃. The increase inmethane production was observed when VCl₃ was added to the cultureswhereby the vanadium stimulated methanogens could utilize formate as thecarbon and energy source, thereby inducing methane production. The dataare consistent with replacement of molybdenum or tungsten with vanadiumas a co-factor for key enzymes (e.g., formate dehydrogenase and/orformyl-MF dehydrogenase) by formate-utilizing methane-producing species.The results are also consistent with the reported metal content of SanJuan basin coal showing several fold more vanadium vs. molybdenum andtungsten available for evolution of vanadium-dependent enzymes. Theseresults are also consistent with the observed decrease in methaneproduction by 2 L-245 with increasing concentrations of molybdate, asuspected antagonist of vanadium-dependent enzymes (see FIG. 3B).

Example 4 Stimulation of Methane Production by Vanadium in CulturesUtilizing Acetate

Acetate is found in coal formation water, is a likely product of coalmatrix degradation and acts a substrate for methanogenic microbes.

The anaerobic reactor system described above in Example 1 containsacetate-utilizing, methane-producing species in the enrichment culture.Methanogens in the culture were previously identified by 16sRNAsequencing.

A 10% vol./vol. inoculum is taken from the anaerobic reactor systemdescribed above in Example 1, and the effect of vanadium on methaneproduction by methanogens utilizing acetate as a carbon and energysource is determined.

Cultures, established with DM media (prepared as described above)containing varying concentrations of acetate as the sole carbon andenergy source, are sub-cultured with the same media (in the absence ofvanadium) or media supplemented with either 5 or 10 μM VCl₃. Methaneproduction is measured after 48 hours of growth at 50° C.

Example 5 Stimulation of Methane Production by Vanadium in Culturesusing Various Other Substrates

Hydrogen, butyrate, propionate and CO₂ are found in coal formationwater, are likely products of coal matrix degradation and can act asubstrate for methanogenic microbes. Hydrogen, butyrate, propionate andCO₂ are found in coal formation water, are likely products of coalmatrix degradation and can act a substrate for methanogenic microbes.

The anaerobic reactor system described above in Example 1 containsmethane-producing species in the enrichment culture. Methanogens in theculture were previously identified by 16sRNA sequencing.

A 10% vol./vol. inoculum is taken from the anaerobic reactor systemdescribed above in Example 1, and the effect of vanadium on methaneproduction by methanogens utilizing hydrogen, butyrate, propionateand/or CO₂ as a carbon and energy source is determined.

Cultures, established with DM media (prepared as described above)containing varying concentrations of hydrogen, butyrate, propionate orCO₂ as the sole carbon and energy source, are sub-cultured with the samemedia (in the absence of vanadium) or media supplemented with either 5or 10 μM VCl₃. Methane production is measured after 48 hours of growthat 50° C.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that elements of the embodimentsdescribed herein can be combined to make additional embodiments andvarious modifications may be made without departing from the spirit andscope of the invention. Accordingly, other embodiments, alternatives andequivalents are within the scope of the invention as described andclaimed herein.

Headings within the application are solely for the convenience of thereader, and do not limit in any way the scope of the invention or itsembodiments.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A method of stimulating production of a metabolic product withenhanced hydrogen content from a hydrocarbon material, comprisingcontacting said hydrocarbon material with a composition comprisingsulfur, an oxyanion of sulfur or vanadium.
 2. The method of claim 1,wherein said metabolic product with enhanced hydrogen content ismethane.
 3. The method of claim 1, wherein said hydrocarbon materialcomprises coal, oil, kerogen, peat, heavy oil, oil shale, tar sands, oilsands, bitumen or tar.
 4. The method of claim 1, wherein said oxyanionof sulfur is thiosulfate, sulfuric acid, disulfuric acid,peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid,thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid orpolythionic acid.
 5. The method of claim 1, wherein said oxyanion ofsulfur is thiosulfate.
 6. The method of claim 1, wherein said vanadiumis vanadium (III) chloride (VCl₃), vanadium (II) chloride (VCl₂), orvanadium (I) chloride (VCl).
 7. A method of stimulating production ofmethane from a hydrocarbon material, comprising contacting saidhydrocarbon material with a composition comprising sulfur, an oxyanionof sulfur or vanadium.
 8. The method of claim 7, wherein saidhydrocarbon material comprises coal, oil, kerogen, peat, heavy oil, oilshale, tar sands, oil sands, bitumen or tar.
 9. The method of claim 7,wherein said oxyanion of sulfur is thiosulfate, sulfuric acid,disulfuric acid, peroxymonosulfuric acid, peroxydisulfuric acid,dithionic acid, thiosulfuric acid, disulfurous acid, sulfurous acid,dithionus acid or polythionic acid.
 10. The method of claim 7, whereinsaid oxyanion of sulfur is thiosulfate.
 11. The method of claim 7,wherein said vanadium is vanadium (III) chloride (VCl₃), vanadium (II)chloride (VCl₂), or vanadium (I) chloride (VCl).
 12. The method of claim7, wherein contacting comprises injection of an aqueous compositioncomprising sulfur, an oxyanion of sulfur or vanadium into a subterraneanformation containing said hydrocarbon material.
 13. The method of claim7, further comprising contacting said hydrocarbon material with acomposition comprising one or more microorganisms, wherein at least oneof said microorganisms is a methanogen.
 14. The method of claim 13,wherein the one or more microorganisms comprise at least one speciesthat is not indigenous to the hydrocarbon material.
 15. The method ofclaim 7, wherein said sulfur, oxyanion of sulfur, or vanadium stimulatesmicroorganisms to metabolize carbonaceous material into methane.
 16. Amethod of stimulating methane production from coal, comprisingcontacting said coal with a composition comprising sulfur, an oxyanionof sulfur, or vanadium.
 17. The method of claim 16, wherein contactingcomprises injection of an aqueous composition comprising sulfur, anoxyanion of sulfur or vanadium into a subterranean coal formation. 18.The method of claim 16, further comprising contacting the coal with acomposition comprising one or more microorganisms, wherein at least oneof same microorganisms is a methanogen.
 19. The method of claim 18,wherein the one or more microorganisms comprise at least one speciesthat is not indigenous to the coal formation where the coal is presentor extracted from.
 20. The method of claim 16, wherein said sulfur,oxyanion of sulfur or vanadium stimulates microorganisms to metabolizecarbonaceous material into methane.
 21. The method of claim 16, whereinsaid oxyanion of sulfur is thiosulfate, sulfuric acid, disulfuric acid,peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid,thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid orpolythionic acid.
 22. The method of claim 16, wherein said oxyanion ofsulfur is thiosulfate.
 23. The method of claim 16, wherein said vanadiumis vanadium (III) chloride (VCl₃), vanadium (II) chloride (VCl₂), orvanadium (I) chloride (VCl).